Generation and Homing of iPSC-Derived Hematopoietic Cells In Vivo

Extensive investigations by dozens of laboratories around the world over the past decade have failed to develop a reproducible and reliable way to differentiate human pluripotent stem cells such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs) into hematopoietic stem cells (HSCs) that are functional both in vivo and in vitro. However, in this issue of Molecular Therapy, Suzuki et al. describe the use of iPSC-derived teratomas as in vivo bioreactors mimicking early embryos for the generation of engraftable HSC-like cells.¹ The iPSC-derived HSC-like cells were shown to be present within the teratoma as well as within the bone marrow (BM) of teratoma-bearing mice. The teratoma-derived HSCs could also engraft and repopulate multiple hematopoietic lineages in mice following subsequent transplantation. These findings provide an important breakthrough in the generation of HSCs with in vivo engraftment capability from iPSCs,^1,2 arguably the first evidence of in vivo biological function by iPSC-derived progeny cells.

There have been extensive attempts to develop methods for the efficient differentiation of PSCs—such as ESCs and iPSCs—into HSCs, which are defined by both the capacity for long-term engraftment in a recipient host and the ability to differentiate into both lymphoid and myeloid cell lineages. The predominant methods to derive hematopoietic cells from PSCs to date are either through a step of cell aggregation called embryoid body formation mimicking early embryos or by coculture with stromal cells such as OP9, a mouse mesenchymal cell line. Using these methods, various hematopoietic cell types have been derived from both mouse and human PSCs, including myeloid lineages and limited lymphoid lineages. However, hematopoietic cells derived in vitro from ESCs or iPSCs typically resemble embryonic or more primitive hematopoietic cells and lack robust in vivo engraftment and repopulation activity following transplantation. Genetic manipulation of mouse ESCs such as forced overexpression of HoxB4 or other transcriptional factors can produce transplantable mouse HSCs,³ although this strategy skews the resulting cells toward the myeloid lineage. HoxB4 overexpression has been shown to enhance the growth of myeloid progenitor cells from human ESCs but is ineffective at promoting HSC-like cells with engrafting activities.^4,5

A teratoma—a benign tumor generated following transplantation of a PSC population into an animal—contains a variety of tissues differentiated from each of the three embryonic germ layers. Teratoma formation is a standard in vivo assay to demonstrate pluripotency of a given stem cell and remains the only in vivo assay for human PSCs.^6,7 The unique cellular complexity of a teratoma, with various cell types including both seed (progenitor) and niche cells, has led to the hypothesis that teratomas may provide an adequate microenvironment for hematopoietic differentiation from PSCs. Although teratomas contain PSC-derived donor cells coexisting with host vascular and hematopoietic cells, there has been no definitive evidence that they can provide an environment conducive to the derivation of engraftable HSCs.

Suzuki et al. first generated teratomas in immunodeficient mice from murine iPSCs derived from wild-type green fluorescent protein (GFP)–transgenic mice or Lnk^–/– GFP–transgenic mice. Lnk^–/– mice were used because they have been shown to overproduce HSCs.⁸ Then the authors further enhanced the outcome toward hematopoietic differentiation by coinjecting OP9 stromal cells with PSCs and supplying two hematopoietic cytokines (stem cell factor and thrombopoietin) through an implanted micro-osmotic pump. After 8–10 weeks, GFP⁺ donor hematopoietic cells expressing the CD45 pan-hematopoietic antigen were found in peripheral blood (PB) of recipient mice. In addition, c-Kit⁺ Sca-1⁺ lineage–negative cells characteristic of mouse HSCs were found in the BM of teratoma-bearing mice and were indeed shown by in vitro assays to be capable of forming hematopoietic colonies. Transplantation of mouse iPSC-derived GFP⁺ BM cells from the teratoma-bearing mice to primary- and secondary-recipient mice gave rise to multilineage hematopoietic reconstitution in vivo. Overall, the data suggest that some of the GFP-labeled iPSCs differentiated into HSC-like cells within the teratoma environment and that these cells were subsequently able to migrate to the BM, where they adopted properties of bona fide HSCs (Figure 1).

Figure 1.

Generation and characterization of iPSC-derived hematopoietic cells in vivo during teratoma formation. Human or mouse induced pluripotent stem cells (iPSCs) were implanted into immunodeficient mice. Donor-specific hematopoietic cells resembling HSC-like cells and their progeny were found within teratoma and in bone marrow. The isolated iPSC-derived HSC-like cells were examined in (a) in vitro assays and (b) an in vivo assay for testing HSC activities in an irradiated mouse to generate both myeloid and lymphoid cells in peripheral blood. Modified from ref. 1.

The authors extended their findings to test the feasibility of using these iPSC-derived hematopoietic cells for therapeutic correction using a combined gene and cell therapy strategy. Mouse iPSCs from X-linked severe combined immunodeficient (X-SCID) mice were generated and then genetically modified to express a functional copy of the γc gene that is missing in these mice, thereby leading to an immunodeficient phenotype. Following injection of the modified iPSCs into X-SCID mice to create a teratoma, the authors were able to detect mouse CD4⁺, CD8⁺, and naive (CD8⁺CD62L⁺) T cells derived from the functionally corrected iPSCs. The data demonstrate the feasibility of a strategy to treat X-SCID disease by deriving hematopoietic progenitor cells through expansion within the teratomas–OP9–cytokine environment.

The group also examined human iPSCs (hiPSCs) for the capacity for teratoma-mediated hematopoiesis. The authors detected cells expressing human CD45 hematopoietic cell markers in both the PB and BM of mice bearing teratomas derived from hiPSCs. Interestingly, hiPSC–derived hematopoietic cells that formed erythroid colony–forming cells expressed both types of β-globin genes (i.e., adult and fetal type). When human CD45⁺CD34⁺ cells isolated from BM of the teratoma-bearing mice were transplanted to new recipient mice, multilineage repopulation was observed, including the generation of human glycophorin A⁺ (erythroid) and CD3⁺ (T lymphoid) cells. Therefore, teratoma-derived HSC-like cells meet the two essential requirements for HSCs: sustained engraftment and the capacity to generate multiple hematopoietic lineages.

Recently, Amabile et al. independently reported similar results, showing that hiPSC-derived teratomas can support in vivo generation of transplantable HSC-like cells as well as functional B and T cells.² There are similarities and differences between the two studies. Both made use of coinjected OP9 stromal cells during teratoma formation. Whereas Suzuki et al. used OP9 cells combined with cytokine supplementation through an osmotic pump,¹ Amabile et al. used genetically modified OP9 cells that constitutionally expressed the Wnt3a gene, a growth factor critical for hematopoiesis.² Amabile et al. observed more extensive lymphoid cell reconstitution than did Suzuki et al. In addition, Suzuki et al. were able to demonstrate that their teratoma-derived HPCs gave rise to adult globin gene expression in differentiated erythroid cells, one of the most definitive markers of definitive hematopoiesis; this was not observed by Amabile et al. It remains to be determined whether the iPSC teratoma–derived hematopoietic cells generated in these two studies possess a potential for differentiation into full myeloid and lymphoid lineages similar to that of postnatal HSCs. Nonetheless, the authors showed conclusive evidence that both murine and human iPSCs undergo hematopoietic differentiation during teratoma formation. The in vivo engraftment capacity suggests that cells resembling functional HSCs were generated that could home to the host BM. However, these findings constitute only the first step on a long journey toward therapeutic application. First, it is unlikely that it will be possible to generate sufficient numbers of transplantable HSCs following teratoma formation in a (large, immunodeficient) animal. Second, it is likely that contamination by both host cells and residual undifferentiated or malignant iPSCs in the derived HSC cell product could preclude their clinical use.

Nonetheless, these studies offer proof of principle and have important implications for the study of early human hematopoiesis. Because human HSC-like cells able to give rise to definitive hematopoiesis were found within teratomas and were shown to be derived from PSCs, we now have available a new model system with which to study the developmental steps in the progression from a PSC to an HSC.

In summary, the use of teratomas as an in vivo bioreactor provides a new method for the derivation of HSCs from PSCs and a new model system for studying the niches required for hematopoietic differentiation. These two studies further complement a recent report that showed that the RUNX1a transgene enhanced in vitro hematopoiesis and in vivo transplantation of human PSC-derived hematopoietic cells.⁹ These novel and complementary approaches renew our enthusiasm to continue a journey of developing better gene and cell therapies by using patient-specific iPSCs to derive HSCs.